Biomedical subjects
M Zuccotti
Publications and source records attributed to M Zuccotti.
Fetal erythroblast isolation up to purity from cord blood and their culture in vitro.
BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.
Gene expression and chromatin organization during mouse oocyte growth.
Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.
Timing of gene expression and oolemma localization of mouse alpha6 and beta1 integrin subunits during oogenesis.
The sperm antigen fertilin alpha/beta and the integrin complex alpha6beta1 present on the oolemma are two of the most promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach the size of 25-30 microm in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine the timing of (1) gene expression of alpha6 and beta1 integrins and (2) localization of these integrin subunits on the plasma membrane in primordial germ cells and in oocytes during oogenesis. We found that both alpha6 and beta1 genes are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p. c., during oocyte growth, and in ovulated eggs. The alternatively spliced isoform alpha6B is expressed from 10.5 d.p.c., whereas alpha6A begins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants. In situ immunodetection of alpha6 or beta1 shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5 d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals and only when oocytes during growth reach the size of about 25-30 microm in diameter does the fluorescence become homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an involvement of alpha6 and beta1 integrins in sperm-egg fusion.
Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.
Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.
Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging.
Two forms of oocytes termed SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus) differing for the spatial distribution of nuclear and nucleolar-associated chromatin have been described within the antral compartment of the ovary of a number of mammals. The biological significance of these two kind of oocytes is as yet not completely clear. In previous studies we have shown that prior to ovulation, mouse SN oocytes isolated from the antral compartment, matured and fertilized in vitro have a far better meiotic and developmental competence than NSN oocytes. Immediately after ovulation SN and NSN oocytes remaining in the antral compartment do not develop beyond the 2-cell stage. To further examine the correlation between chromatin distribution and meiotic competence of mouse antral oocytes, in the present study we have analyzed chromosome segregation at the first meiotic division in antral (SN and NSN) and in ovulated oocytes. SN and NSN oocytes were isolated before (48 h post PMSG injection) or after (15 h post-hCG injection) ovulation from ovaries of females of increasing age, they were cultured in vitro to metaphase II, and their aneuploidy rate was examined. Comparison of data obtained before and after ovulation highlights two main points: 1. Following ovulation a statistically significant increase of aneuploidy is observed in antral oocytes in most age groups and it is attributable to SN oocytes. 2. The aneuploidy rate of ovulated oocytes does not increase during female aging. We have found a correlation between chromatin distribution, hormonal status, and the incidence of aneuploidy during the oocyte first meiotic division.
A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility.
Premature ovarian failure (POF) is a defect of ovarian development and is characterized by primary or secondary amenorrhea, with elevated levels of serum gonadotropins, or by early menopause. The disorder has been attributed to various causes, including rearrangements of a large "critical region" in the long arm of the X chromosome. Here we report identification, in a family with POF, of a gene that is disrupted by a breakpoint. The gene is the human homologue of the Drosophila melanogaster diaphanous gene; mutated alleles of this gene affect spermatogenesis or oogenesis and lead to sterility. The protein (DIA) encoded by the human gene (DIA) is the first human member of the growing FH1/FH2 protein family. Members of this protein family affect cytokinesis and other actin-mediated morphogenetic processes that are required in early steps of development. We propose that the human DIA gene is one of the genes responsible for POF and that it affects the cell divisions that lead to ovarian follicle formation.
Meiotic and developmental competence of mouse antral oocytes.
Mouse antral oocytes show two different patterns of chromatin organization, defining oocytes with or without chromatin surrounding the nucleolus (SN: surrounded nucleus; NSN: not surrounded nucleus). We have previously shown that upon injection of eCG, NSN antral oocytes shift towards the SN kind of chromatin organization. We hypothesized that these newly formed SN oocytes were those that would have been ovulated after an ovulatory stimulus. The main objective of this study was to investigate the meiotic and developmental competence of these two types of oocytes. SN and NSN antral oocytes were isolated after i.p. administration of eCG + hCG or eCG-only, in vitro-cultured until completion of metaphase II, and inseminated with capacitated spermatozoa; and their development to the 4-cell stage was examined. This study demonstrates 1) that SN and NSN oocytes isolated after injection of eCG + hCG are capable of embryonic development, but not beyond the 2-cell stage; and 2) that SN and NSN oocytes isolated after injection of eCG-only are capable of developing to the 2-cell stage, but a significantly higher number (11.9%) of SN oocytes than NSN oocytes (1.5%) reach the 4-cell stage. SN- and NSN-like oocytes have also been described in the antral compartment of human, rat, monkey, pig, and bovine ovaries. The findings reported in this paper may contribute to improved procedures for in vitro fertilization of humans and farm animals.
Six-year-old archival chromosome preparations are still good biological reagents for repeated primed in situ labelling (rPRINS).
Six-year-old chromosome preparations of Eulemur coronatus were used for in situ localization of highly repetitive DNA sequences. The application of the repeated primed in situ labelling (rPRINS) technique allowed the detection of positive signals whereas conventional fluorescence in situ hybridization gave negative results on the same archival preparations. This paper reveals the possibility of rescuing archival chromosome preparations for both clinical and comparative cytogenetics. Moreover, the chromosomal localization of the ECO-SAT 503 bp highly repetitive DNA sequences were found to localize in the pericentromeric region of every chromosome, with the exception of chromosome 9.
XIST expression in human oocytes and preimplantation embryos.
During mouse preimplantation development, the exclusive expression of the Xist gene from the paternally inherited allele is thought to play a role in the inactivation of the paternally inherited X chromosome in the extra-embryonic cell lineages of the developing female embryo. Recently, inactivation of the paternally inherited X chromosome has also been shown to occur in the extraembryonic cell lineages of the human female conceptus. In this paper, we determine whether the pattern of XIST expression in human preimplantation embryos is similarly correlated with paternal X inactivation. We developed procedures sensitive to the single cell, for the simultaneous analysis of XIST and HPRT expression and of sexing, initially using human fibroblast cells. Application of these procedures to human cleavage-stage embryos derived by in vitro fertilization revealed a pattern of XIST expression different from that in the mouse. Transcripts of the XIST gene were detected as early as the 1-cell zygote and, with increasing efficiency, through to the 8-cell stage of preimplantation development. In addition, transcripts of XIST were detected in both male (hence from the maternally inherited allele) and female preimplantation embryos. This pattern of expression is not consistent with a role for the early expression of the XIST gene in the choice of paternal X inactivation in the extraembryonic cell lineages of the developing human embryo.
Genome composition in Venezuelan spiny-rats of the genus Proechimys(Rodentia, Echimyidae). I. Genome size, C-heterochromatin and repetitive DNAs in situ hybridization patterns.
The genome sizes of the Venezuelan spiny-rats Proechimys guairae guairae (2n = 48) and P. trinitatis (2n = 62) were evaluated and proved to be 12.5 +/- 0.5 pg and 12.6 +/- 0.3 pg respectively, the highest so far recorded among mammals; also the C-heterochromatin (32.7%, Coefficient of Variation [CV] 3.8 and 35.8%, CV 4.4) and GC (44.2%, CV 2.7 and 43.6%, CV 2.9) contents are very high. Highly repetitive (rep) DNAs were isolated from restriction enzyme digested genomic DNAs of both species. The intra- and inter-specific chromosomal allocations of these rep DNAs were analyzed by direct and cross-hybridizations. Results show that the two genomes harbour several rep DNA families which show both species-specificity and interspecific relatedness in their in situ hybridization patterns. The rep DNA families show an equilocal distribution at both the pericentromeric areas of all chromosomes and in the whole arms of two pairs of the uniarmed group, suggesting co-evolution of the rep DNAs. P. g. guairae BamHI digested DNA, when cloned and sequenced, proved to consist of a long "composite" unit (1,239 bp) containing two copies of each of 75-bp and 110-bp internal subrepeats. Karyotype restructuring between P. g. guairae and P. trinitatis, mainly due to Robertsonian changes, was accompanied by slight intra- and intergenomic movements of the putative satellite DNA families within stable genome sizes and C-heterochromatin contents. We discuss the findings obtained in Proechimys in the light of those regarding the kangaroo rat, the pocket gopher and the house mouse; they support the idea that karyotype restructuring could be the expression of molecular driven events of rep DNA amplification and homogenisation through non-homologous chromosomes.
The mouse Xist gene: a model for studying the gametic imprinting phenomenon.
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Chromatin organization during mouse oocyte growth.
We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I-III, 10-40 microns in diameter: 100% NSN vs. 0% SN; class VII 70-80 microns in diameter: 47.3% NSN vs. 52.3 SN, in 4-6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation.
Methylation of the mouse Xist gene in sperm and eggs correlates with imprinted Xist expression and paternal X-inactivation.
Preferential paternal X-inactivation in the extra-embryonic tissues of the female mouse embryo is correlated with imprinted expression of the paternal allele of the Xist gene in pre-implantation development. Here we examine 11 CpG sites in Xist to determine whether differential methylation might be the molecular basis for imprinting. We find that three sites in the promoter region are methylated in eggs but not in sperm and that this differential methylation is maintained to the blastocyst stage when the paternal X-inactivation occurs. This is the first example of a primary gametic methylation imprint governing differential expression of parental alleles in pre-implantation embryos.
Development and loss of the ability of mouse oolemma to fuse with spermatozoa.
To further our knowledge on the mechanisms and molecules involved in mouse sperm-oocyte plasma membrane interaction, experiments were carried out to determine the stage during oogenesis at which an oocyte acquires the capacity to fuse with acrosome-reacted sperm. Zona-free oocytes 10 microns in diameter do not fuse with sperm. Oolemma fusibility is first acquired when the oocyte reaches about 20 microns in diameter. Fusibility is maintained even after fertilisation has occurred and is lost completely by the 4-cell stage.
Polymyositis-like syndrome in hypothyroidism: report of two cases.
An increase of muscle enzymes in hypothyroidism has sometimes been correlated with a polymyositis-like syndrome and hypothyroid patients have been misdiagnosed and mismanaged as suffering from polymyositis. Actually, muscle symptoms, such as aches and pain, stiffness, weakness and cramps or, more rarely, hypertrophy, are observed in hypothyroidism and increased serum muscle enzyme values, particularly the level of creatine phosphokinase (CPK), indeed seem to suggest polymyositis. The muscular groups most commonly affected by the above mentioned symptomatology are those of the shoulder and pelvic girdles. In this report two hypothyroid patients complaining of muscle symptoms, whose serum muscle enzymes were particularly elevated, are described. In the first case the patient had been suffering from pain and weakness of the thenar eminence for about 4 months. The clinical features suggested a diagnosis of Carpal Tunnel Syndrome, but thyroid function tests revealed primary hypothyroidism. In the second case the patient had been afflicted with muscular weakness of the shoulder girdle for 2 months and was unable to keep his arms raised over his head. A study of thyroid function demonstrated a picture of hypothyroidism. Both patients were treated with L-thyroxine and in a relatively short time biochemical parameters improved remarkably, and the symptoms disappeared. The hypothesis that a muscular effort of long duration by hypothyroid patients may have been responsible for the muscular damage and the symptoms may explain why only a few hypothyroid patients develop a clinical picture similar to polymyositis.